The present invention relates to a thermostable collagen-decomposing enzyme produced by a new microorganism, and said new microorganism and a method for production of said enzyme by said microorganism. Concretely, the present invention relates to a novel thermostable collagen-decomposing enzyme having the highest reactivity (substrate specificity) to collagen, produced by a novel microorganism of the genus Bacillus, said novel microorganism and a process for producing of said enzyme by said microorganism.
Collagen-decomposing and gelatin-decomposing enzymes have been widely used in industry. For example, collagen peptides, which are hydrolytic products from collagen by these enzymes, are useful as material for cosmetics, because of their interesting physiological activities such as moisture-keeping effects or immunity-activation activity. Therefore, collagen peptides are widely used for medical and cosmetic purposes. Further, gelatin, which is a denaturated form of collagen, is used as the coating material for photograph films, and gelatin-decomposing enzymes are used for the recycling of the photograph and X-ray films. Many kinds of proteases are known to decompose difficult. For hydrolysis of collagen, specific metal proteases, named xe2x80x9ccollagenasexe2x80x9d, should be used.
Recently, many attempts have been made for the effective use of the organic wastes. For instance, the composing of garbage wastes is one concrete example of the biorecycling of organic wastes. More than 30% of animal protein are composed of collagen, therefore, garbage wastes produced from daily kitchen activities in houses and restaurants and the wastes from meat processing factories should contain large quantities of collagen.
Because of specific, highly ordered structure, collagen is generally insoluble in water and difficult to be decomposed, and therefore degradation of collagen proceeds very slowly during composing. Most part of unusable portions produced from livestock industries are composed of collagen, and, therefore, are treated by incineration, causing problems such as anathermal of the earth or the generation of carbon dioxide or dioxin which cause the air pollution.
These problems must be solved from the view point to make an effective use of materials.
It is well known that the temperature of the organic wastes raises to 50-65xc2x0 C. or higher during the composing process. Therefore, if thermostable enzymes or thermophilic microorganisms which are active even under such high-temperature composing conditions are used, the composing of organic waste should proceed more effectively.
Nowadays, industrial collagenases are those from microorganisms (bacteria), and as a concrete example, an actinomycetous collagenase of the genus Streptomyces can be mentioned. Other microbial collagenases are also known; for instance, collagenases from Clostridium hystolyticum (Biochemistry 1984, 23, 3077-3085) and Cytophaga sp. (Biosci, Biotech, Biochem., 1993, 57, 1894-1898) are the concrete examples.
Concerning the example of enzyme which is industrially used, it is necessary for the enzyme to be thermostable from the view point of treating speed and the subject to be treated.
However, all known collagenases are of mesophilic origin and lacks of thermostability, and these circumstances hampers their efficient industrial applications. Until now, a collagen-decomposing enzyme with sufficient thermostability (having high optimum temperature) for the industrial applications has not yet been developed.
Usually, it is difficult to use collagen in an industrial scale because there is no thermostable enzyme to act effectively in an industrial scale. Therefore, it is obvious that above mentioned problem can be solved perfectly, if an enzyme having high activity to collagen is developed.
As mentioned above, the object of this invention is to find out a thermostable collagen-decomposing enzyme.
The inventors of this invention have carried out an intensive study to find a microorganism producing a thermostable collagen-decomposing enzyme in nature, and have found a promising thermophilic bacterium belonging to the genus Bacillus genus that produces said enzyme in the soil of Sendai, Japan and accomplished the present invention.
The microorganism, which is used to produce a thermostable collagen-decomposing enzyme of this invention, belongs to the genus Bacillus, and is termed strain NTAP-1. This strain has been deposited according to the requirement of deposit based on Budapest treaty in the Biotic Technology Industries Institute of the Agency of Industrial Science of Technology belonging to the Ministry of International Trade and Industry Japan and accepted by the accept number of FERM BP-6926 on Nov. 1, 1999 Biotic Technology Industries Institute, Agency of Industrial Science and Technology 1-3, Higashi 1-chome Tsukuba-shi, Ibaraki-ken 305-0046 JAPAN. (This strain is originally deposited on Aug. 27, 1999 under accession number FERM P-17535.) (in the specification, this strain is shortened only as  less than NTAP-1 strain greater than )
The inventors of this invention have found out that the industrially useful enzyme can be obtained by the use of this strain, and the obtained enzyme can be used as the catalyst for bioconversion.
The first important point of this invention is a thermostable collagen-decomposing enzyme obtained by the microorganism having a thermostable collagen-decomposing activity and belonging to the genus Bacillus, which is characterized by the following features; (1) the bacterium is Gram-negative or Gram-indefinite, (2) the bacterium has a spore forming ability, (3) the bacterium is motile, (4) the bacterium grows at 70xc2x0 C., does not grow at 30xc2x0 C. or 80xc2x0 C. and grows at pH 5, does not grow at pH 7, (6) the bacterium is rod-shaped, (7) the bacterium is negative to catalase, (8) the bacterium is negative to oxidase, (9) the bacterium is negative to O/F test, (10) the bacterium has acetoin producing activity and (11) the bacterium has gelatin decomposition activity. Accordingly, an excellent action and effect which can be used for the decomposition of collagen at 70xc2x0 C. or lower temperatures can be expected.
Desirably, said thermostable collagen-decomposing enzyme of this invention is characterized by the following features: (1) the enzyme can far more effectively hydrolyze collagen and gelatin than casein and albumin, (2) optimum reaction pH is between pH 3.5 and 4.5, (3) optimum reaction temperature is between 65xc2x0 C. and 70xc2x0 C., (4) the enzyme retains more than 60% of its original activity after heat treatment at 60xc2x0 C. and pH 6.0 for 4 hours, (5) the enzyme is stable between pH 3 to 6 and (6) molecular weight of the enzyme estimated by SDS-polyacrylamide gel electrophoresis is approximately 46,000. And more desirably, said thermostable collagen-decomposing enzyme of this invention is produced by the microorganism belonging to the genus Bacillus or Bacillus sp. strain NTAP-1.
The second important point of this invention is a producing method of the thermostable collagen-decomposing enzyme comprising, using a microorganism which has following features, that is, by the following features: (1) the bacterium is Gram-negative or Gram-indefinite, (2) the bacterium has a spore forming ability, (3) the bacterium is motile, (4) the bacterium grows at 70xc2x0 C., does not grow at 30xc2x0 C. or 80xc2x0 C. and grows at pH 5, does not grow at pH 7, (6) the bacterium is rod-shaped, (7) the bacterium is negative to catalase, (8) the bacterium is negative to oxidase, (9) the bacterium is negative to O/F test, (10) the bacterium has acetoin producing activity and (11) the bacterium has gelatin decomposition activity, purifying and accumulating the thermostable collagen-decomposing enzyme which has following features: (1) the enzyme can far more effectively hydrolyze collagen and gelatin than casein and albumin, (2) optimum reaction pH is between pH 3.5 and 4.5, (3) optimum reaction temperature is between 65xc2x0 C. and 70xc2x0 C., (4) the enzyme retains more than 60% of its original activity after heat treatment at 60xc2x0 C. and pH 6.0 for 4 hours, (5) the enzyme is stable between pH 3 to 6 and (6) molecular weight of the enzyme estimated by SDS-polyacrylamide gel electrophoresis is approximately 46,000, in a culture medium and by collecting it.
Desirably, the producing method of said thermostable collagen-decomposing enzyme, wherein the microorganism belonging to the genus Bacillus is the Bacillus genus bacteria NTAP-1 strain.
The third important point of this invention is a new developed microorganism belonging to a Bacillus genus, which produces said thermostable collagen-decomposing enzyme, desirably, said microorganism is the strain titled as Bacillus sp. NTAP-1 and have deposited according to the requirement of deposit based on Budapest treaty in the Biotic Technology Industries Institute of the Agency of Industrial Science of Technology belonging to the Ministry of Intentional Trade and Industry Japan and accepted by the accession number FERM BP-6926 on Nov. 1, 1999 Biotic Technology Industries Institute, Agency of Industrial Science and Technology 1-3, Higashi 1-chome Tsukuba-shi, Ibaraki-ken 305-0046 JAPAN. (This strain is originally deposited on Aug. 27, 1999 under accession number FERM P-17535.).
The inventors of this invention have found that among the microorganism belonging to Bacillus genus there is a novel microorganism which produces thermostable collagen-decomposing enzyme, and have accomplished the present invention.